In the development research of antibodies for treatment, a hybridoma technology capable of producing monoclonal antibodies was developed about 35 years ago. Additionally, chimeric/humanized antibodies overcoming immunogenicity of mouse antibodies (HAMA; Human anti-mouse antibody response) initially received its clinical approval in 1997. In addition, complete human antibody, Humira received approval in 2003. Furthermore, in order to increase treatment efficacy, the research on bispecific antibodies, antibody drug conjugation (ADC), and long-lasting antibodies improving heavy-chain constant region (Fc) has been actively conducted.
In the case of antibodies for treating a solid tumor, during a process where the antibodies are transferred to tumor tissues, the amount of antibodies transferred to the tumor tissues in actual bodies of human beings is merely 0.01 to 0.001% of the amount injected by various barriers, which means that the treatment effect of antibodies is very limited (Thurber et al. 2008). Accordingly, the development of antibody technology allowing the antibodies to be selectively accumulated in the tumor tissues and to have high permeability into the tumor tissues may increase the treatment effect of antibodies and thus is very important.
There are two major reasons why the antibodies are not well permeated into tissues: 1) intrinsic properties of antibodies (size, antigen-binding properties, etc.) (Thurber and Dane Wittrup, 2012) and 2) fine physiological properties of tumor tissues which are different from normal tissues (Jain and Stylianopoulos, 2010).
Since antibodies are big molecules of 150 kDa consisting of 12 domains, the antibodies in the blood are difficult to be transferred to the tumor tissues through diffusion or convection (Baker et al. 2008). Thus, among the research conducted to solve this, there was an attempt to administer only the domain which binds to an antigen of antibody. In the case of single-chain antibody fragment (scFv, 30 kDa) and heavy-chain variable region (VHH, 14 kDa), they permeated into the tumor tissues much more than the antibodies themselves. However, as their size decrease, a great deal thereof come out through the kidney, and thereby a half-life gets shorter. Thus, efficacy of antibodies has not been significantly improved (Behr et al. 1998).
Another reason why the antibodies are not distributed in a large amount in the tissues is an antigen binding capacity of antibodies. The antibodies for treating the solid tumor are over-expressed in a tumor-associated antigen, or in a tumor, and have a high affinity to a target which is important for the tumor's growth. Even when the antibodies reach the tissues where a specific antigen is present, in the tumor tissues consisting of cells with a great amount of antigen expression, the antibodies are stayed in the antigen while binding to the antigen, due to their high affinity (Lee and Tannock, 2010). Also, after the binding, the antibodies penetrated into the cells along with antigens (Endocytosis) and are lysed. Consequently, the antibodies cannot exert their anti-cancer effects. In order to overcome this, the research for adjusting affinity or lengthening half-life has been processed (Dennis et al. 2007).
Physiological properties of tumor tissues which prevent the antibodies from being permeated and distributed in the tumor tissues may be broadly classified into 4 cases, which are endothelial barrier, high interstitial fluid pressure, stromal impediment, and epithelial barrier.
For the endothelial barrier, the tumor over-expresses and secretes factors (pro-angiogenic factor) which promote the growth of vascular endothelial cells located around blood vessel in order to receive a great deal of nutrients according to the tumor's rapid speed of growth. Accordingly, a large amount of new blood vessels are produced in an uneven manner, which leads to a decrease in the speed of entire blood flow. In order to overcome this, there is a method for increasing extravasation so that therapeutic agents could come out of the blood vessel and be distributed into the tissue. There is a case of enhancing a drug delivery into tumor tissue by co-administering TNF-α and IL02, which are cytokine inflammatory responses related with extravasation, chemical substance promoting extravasation (promoter chemical drug) (Marcucci et al. 2013), iRGD peptide, etc. in combination with therapeutic agents. However, these attempts were difficult to be commercialized and clinically experimented in that it is required to produce two substances of antibodies and extravasation promoter. Additionally, iRGD peptide reached its limit in that it needs to be administered in a great quantity (2 mg/kg or 4 mg/kg) (Sugahara et al. 2010).
High tumor interstitial fluid pressure results from a situation where the pressure difference allowing the drug to be convected from the blood vessel to the tissue is small, or where the fluid pressure of tissue is higher than that of blood. High tumor interstitial fluid pressure is mainly caused due to the accumulation of interstitial fluid pressure in the absence of lymphatic duct in the tumor tissue, unlike in the normal tissue, and contributes to abnormal angiogenesis. In order to overcome this, a method for preventing the operation of a factor promoting the growth of vascular endothelial cell, particularly vascular endothelial cell growth factor-A (VEGF-A), and inhibiting angiogenesis to normalize the blood vessel, or a method for increasing the fluid pressure of blood vessel has been attempted. With regard to the method for increasing the fluid pressure of blood vessel, there was a case where plasma protein albumin was administered in combination with antibodies to increase osmotic pressure of blood vessel, thereby improving delivery effect of antibodies to the tumor tissue (Hofmann et al. 2009).
The stromal impediment, which is an extracellular matrix barrier met when the antibodies come out to micro-vessels and are convected to the tissue, mainly consists of collagen and hyaluronan. The extracellular matrix greatly affects the shape of tumor. Accordingly, there is a big difference between the area where the drug is well distributed and the area where the drug is not well distributed, so drug distribution becomes uneven. Additionally, as an amount of expression of extracellular matrix increases, the tumor interstitial fluid pressure due to a high cell density with solid tumor stress (solid stress) increases. As the method for overcoming this, there is a method for inducing apoptosis of tumor tissue cell to reduce tumor interstitial cell density. Additionally, there was a case increasing the drug delivery effect about two times compared to a control group by processing an enzyme (collagenase) dissolving collagen in the tumor tissue to reduce solid stress (Eikenes et al. 2004).
In the epithelial barrier, intercellular adhesion factors of tumor interstitial epithelial cell densely fill up an intercellular space, and thus they prevent the therapeutic agent from being diffused and convected between the cells. E-cadherin is well known as a main factor of the intercellular adhesion. Since a substance reducing the E-cadherin was found in virus (adenovirus-3), there was a case where only a part (JO-1) with an activity of reducing E-cadherin of cell among proteins constituting the virus was administered in combination with the antibody, thereby increasing an anti-cancer effect of antibody (Beyer et al. 2011).
Considering the methods suggested so far to readily deliver the therapeutic agents to the tumor, most cases simply administer the therapeutic agents in combination with the substance for delivering this therapeutic agents well to the tumor tissue. Particularly, peptide is pharmacokinetics resulting from a small size of molecule, and has very short half-life. Thus, a great amount of peptide needs to be administered to actual patients and the administration needs to be frequently made. Furthermore, since the therapeutic agents and substances for tumor permeation operation need to be produced, respectively, which is an inevitable process during co-administration, its industrial practicability is low. Also, the peptide sequence and protein which do not exist in the natural world are likely to cause immunogenicity. Thus, ideally, it is required to develop a format where the antibody acquires tissue permeability as it is so that the delivery effect of one antibody molecule into the tumor tissue could be increased.
Among the proteins existing in the natural field, the vascular endothelial cell growth factor-A (VEGF-A) is well known for inducing blood spout (extravasation), which is also called as a vascular permeability factor. This action is known as a phenomenon caused by the binding with a vascular endothelial cell growth factor receptor (VEGFR2). Interestingly, in a mutant experiment of the vascular endothelial cell growth factor-A, even if the factor is not combined to the vascular endothelial cell growth factor receptor, vascular permeability increased, which suggests that another receptor of the vascular endothelial cell growth factor-A exists (Stacker et al. 1999). Other professionals of the same age found that this receptor is neuropilin (NRP).
Neuropilin was first found in a Xenopus nervous system. Neuropilin is a transmembrane glycoprotein, and has two types of NRP1 and NRP2. Neuropilin is expressed very weakly in normal cells, whereas is over-expressed in tumor vascular endothelial cells, solid tumor cells, blood tumor cells. Neuropilin operates as a coreceptor of VEGFRs (VEGF receptors) by binding to VEGF family ligands. Especially, NRP1 operates as a coreceptor of VEGFR1, VEGFR2 and VEGFR3, and binds to various VEGF ligands, thereby contributing to angiogenesis, cell survival, migration & adhesion, invasion, etc. in the tumor tissue. In comparison, NRP2 operates as a coreceptor of VEGFR2 and VEGFR3, thereby contributing to lymphangiogenesis and cell adhesion. Additionally, NRP1/NRP2 (NRP1/2) operates as a coreceptor of plexin family receptors to bind to secreted class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F and Sema3G). Since the neuropilin has no domain in functional cells, even if a ligand is binding thereto, the neuropilin has no activity by itself. It is known that signals are transferred through the VEGF receptor, which is the coreceptor, or through the plexin co-receptor. Sema3 binds to neuropilin and the plexin receptor at a ratio of 2:2:2 and operates.
There are cases reported that the operation of neuropilin and coreceptor is inhibited even when the neuropilin alone is targeted. For example, in the case of anti-neuropilin-1 antibody, it is reported that the anti-neuropilin-1 antibody is competitive to bind only to neuropilin-1 with VEGA-A, which is well known for binding to VEGFR2 and neuropilin-1, and has the function of inhibiting angiogenesis, cell survival, migration & adhesion and invasion, which are operations of VEGFR2 (Pan Q et al 2007). In the case of anti-neuropilin-2 antibody, it is reported that anti-neuropilin-2 antibody is competitive to bind to neuropilin-2 with VEGA-C which is well known for binding to VEGFR3 and neuropilin-2 at the same time, and has the function of inhibiting lymphangiogenesis and cell adhesion, which are operations of VEGFR3 (Cant M et al. 2008).
Thus, the present inventors infer a part of minimum Sema3A- or Sema3F-derived peptide with an enhancing effect of vascular endothelial cell permeability by the interaction between the neuropilin and Sema3A or Sema3F, and designed a mutant peptide so as to have a high affinity with the neuropilin. The present inventors designed bivalent shape by converging peptide into a heavy-chain C-terminus of antibody so that the peptide could copy the function of Sema3A/Sema3F operating as a homodimer. As this design is a single molecule fused with antibody-peptide, a unique function of antibody is maintained as it is. Additionally, by promoting the antibody's accumulation into the tumor tissue and permeability into the tumor tissue by the binding to the neuropilin, and additionally inhibiting the operation of neuropilin coreceptor by the neuropilin targeting, a fusion antibody technology with an anti-angiogenesis effect has been developed.